Limits...
Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa

View Article: PubMed Central - PubMed

ABSTRACT

Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.

No MeSH data available.


Related in: MedlinePlus

Structure of PaLpxH.(a) Ribbon diagram of the Pseudomonas aeruginosa LpxH (PaLpxH) shown in rainbow color (P21 crystal with Mn2+). Lipid X is depicted by green sticks and the Mn2+ ions are violet spheres. PaLpxH consists of a catalytic domain of approximately 180 residues (Met1–Leu118 and Val174–Leu240) and a helical insertion domain (HI domain) (α4–α7). (b) Surface representation of the PaLpxH. The structure is colored in red with different density based on the hydrophobicity scale30. (c) Cross-sectional view of the PaLpxH focusing to hydrophobic cavity between the catalytic and HI domains. The 2-acyl chain of the lipid X is deeply buried in the cavity. d. Fo–Fc omit maps superposed with bound lipid X (cut off 3.5 σ).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016852&req=5

f2: Structure of PaLpxH.(a) Ribbon diagram of the Pseudomonas aeruginosa LpxH (PaLpxH) shown in rainbow color (P21 crystal with Mn2+). Lipid X is depicted by green sticks and the Mn2+ ions are violet spheres. PaLpxH consists of a catalytic domain of approximately 180 residues (Met1–Leu118 and Val174–Leu240) and a helical insertion domain (HI domain) (α4–α7). (b) Surface representation of the PaLpxH. The structure is colored in red with different density based on the hydrophobicity scale30. (c) Cross-sectional view of the PaLpxH focusing to hydrophobic cavity between the catalytic and HI domains. The 2-acyl chain of the lipid X is deeply buried in the cavity. d. Fo–Fc omit maps superposed with bound lipid X (cut off 3.5 σ).

Mentions: PaLpxH was crystallized in the presence of Mn2+ ions, which are required for enzymatic activity68. The structure was determined with the multi-wavelength anomalous diffraction method using Mn2+ ions. Two PaLpxH molecules were present in the asymmetric unit of the P21 crystal, and all 239 amino acid residues were unambiguously determined for both molecules (Fig. 2a). Furthermore, the high-resolution electron density (1.65 Å) clearly identified a stoichiometrically bound product molecule (lipid X, 2,3-diacylglucosamine-1-phosphate) and two Mn2+ ions in each PaLpxH molecule (Fig. 2). The lipid X molecule was likely captured in E. coli cells because no substrate molecule (UDP-2,3-diacylglucosamine) or product molecule (lipid X) was added during purification or crystallization. Proteins without Mn2+ ions were also successfully crystallized in two different space groups. A C2 form was obtained with no Mn2+ ions added during crystallization, and a P21 form was obtained by adding EDTA prior to crystallization. These crystals also contained two PaLpxH molecules in the asymmetric unit. The two molecules in the asymmetric unit had similar hydrophobic contacts with each other in all these crystals (see below). No major changes were observed in the protein structures regardless of the presence of Mn2+ ions (Fig. S1), suggesting that Mn2+ ions do not have a major effect on the protein structure.


Crystal structures of the UDP-diacylglucosamine pyrophosphohydrase LpxH from Pseudomonas aeruginosa
Structure of PaLpxH.(a) Ribbon diagram of the Pseudomonas aeruginosa LpxH (PaLpxH) shown in rainbow color (P21 crystal with Mn2+). Lipid X is depicted by green sticks and the Mn2+ ions are violet spheres. PaLpxH consists of a catalytic domain of approximately 180 residues (Met1–Leu118 and Val174–Leu240) and a helical insertion domain (HI domain) (α4–α7). (b) Surface representation of the PaLpxH. The structure is colored in red with different density based on the hydrophobicity scale30. (c) Cross-sectional view of the PaLpxH focusing to hydrophobic cavity between the catalytic and HI domains. The 2-acyl chain of the lipid X is deeply buried in the cavity. d. Fo–Fc omit maps superposed with bound lipid X (cut off 3.5 σ).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016852&req=5

f2: Structure of PaLpxH.(a) Ribbon diagram of the Pseudomonas aeruginosa LpxH (PaLpxH) shown in rainbow color (P21 crystal with Mn2+). Lipid X is depicted by green sticks and the Mn2+ ions are violet spheres. PaLpxH consists of a catalytic domain of approximately 180 residues (Met1–Leu118 and Val174–Leu240) and a helical insertion domain (HI domain) (α4–α7). (b) Surface representation of the PaLpxH. The structure is colored in red with different density based on the hydrophobicity scale30. (c) Cross-sectional view of the PaLpxH focusing to hydrophobic cavity between the catalytic and HI domains. The 2-acyl chain of the lipid X is deeply buried in the cavity. d. Fo–Fc omit maps superposed with bound lipid X (cut off 3.5 σ).
Mentions: PaLpxH was crystallized in the presence of Mn2+ ions, which are required for enzymatic activity68. The structure was determined with the multi-wavelength anomalous diffraction method using Mn2+ ions. Two PaLpxH molecules were present in the asymmetric unit of the P21 crystal, and all 239 amino acid residues were unambiguously determined for both molecules (Fig. 2a). Furthermore, the high-resolution electron density (1.65 Å) clearly identified a stoichiometrically bound product molecule (lipid X, 2,3-diacylglucosamine-1-phosphate) and two Mn2+ ions in each PaLpxH molecule (Fig. 2). The lipid X molecule was likely captured in E. coli cells because no substrate molecule (UDP-2,3-diacylglucosamine) or product molecule (lipid X) was added during purification or crystallization. Proteins without Mn2+ ions were also successfully crystallized in two different space groups. A C2 form was obtained with no Mn2+ ions added during crystallization, and a P21 form was obtained by adding EDTA prior to crystallization. These crystals also contained two PaLpxH molecules in the asymmetric unit. The two molecules in the asymmetric unit had similar hydrophobic contacts with each other in all these crystals (see below). No major changes were observed in the protein structures regardless of the presence of Mn2+ ions (Fig. S1), suggesting that Mn2+ ions do not have a major effect on the protein structure.

View Article: PubMed Central - PubMed

ABSTRACT

Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.

No MeSH data available.


Related in: MedlinePlus